The stereospecific incorporation of fenoprofen into rat hepatocyte and adipocyte triacylglycerols

The formation of triacylglycerols containing fenoprofen was studied in rat isolated adipocytes and hepatocytes incubated with [3H]glycerol and R or S fenoprofen. In both hepatocytes and adipocytes there was a high-affinity enzymatic process for the synthesis of triacylglycerol containing fenoprofen which was stereospecific for the R enantiomer. The apparent Km values for R fenoprofen were 1.0 microM in adipocytes and 2.8 microM in hepatocytes. These results are consistent with the proposed stereospecific formation of R-2-arylpropionyl-CoA thioesters resulting in the stereospecific formation of R-tri-acylglycerol at clinically relevant unbound fenoprofen concentrations. In isolated hepatocytes, but not adipocytes, a second low-affinity enzymatic process for the synthesis of triacylglycerol containing fenoprofen was also observed. However, this process (Km = 3780 microM) occurred at concentrations much higher than those found in man with usual doses.     

In vitro reactivity of carboxylic acid-CoA thioesters with glutathione

The chemical reactivity of acyl-CoA thioesters toward nucleophiles has been demonstrated in several recent studies. Thus, intracellularly formed acyl-CoAs of xenobiotic carboxylic acids may react covalently with endogenous proteins and potentially lead to adverse effects. The purpose of this study was to investigate whether a correlation could be found between the structure of acyl-CoA thioesters and their reactivities toward the tripeptide, glutathione (gamma-Glu-Cys-Gly). The acyl-CoA thioesters of eight carboxylic acids (ibuprofen, clofibric acid, indomethacin, fenbufen, tolmetin, salicylic acid, 2-phenoxypropionic acid, and (4-chloro-2-methyl-phenoxy)acetic acid (MCPA)) were synthesized, and each acyl-CoA (0.5 mM) was incubated with glutathione (5.0 mM) in 0.1 M potassium phosphate (pH 7.4, 37 degrees C). All of the acyl-CoAs reacted with glutathione to form the respective acyl-S-glutathione products, with MCPA-CoA having the highest rate of conjugate formation (120 +/- 10 microM/min) and ibuprofen-CoA having the lowest (1.0 +/- 0.1 microM/min). The relative reactivities of the acyl-CoAs were dependent on the substitution at the carbon atom alpha to the acyl carbon and on the presence of an oxygen atom in a position beta to the acyl carbon and were as follows: phenoxyacetic acid > o-hydroxybenzoic acid--phenoxypropionic acid > arylacetic acid derivatives > 2-methyl-2-phenoxypropionic acid--2-phenylpropionic acid. For each acyl-CoA thioester, the overall hydrolysis rate was determined as the time-dependent formation of parent compound. A linear trend was observed when comparing the reactivities of the acyl-CoAs with glutathione with the corresponding overall hydrolysis rates. Thus, the most reactive compound (MCPA-CoA) was also the compound with the highest rate of hydrolysis and the least reactive compounds (ibuprofen-CoA, clofibryl-CoA) were also the compounds least susceptible to hydrolysis.     

Inhibition of rat peroxisomal palmitoyl-CoA ligase by xenobiotic carboxylic acids.

ATP-dependent coenzyme A (CoA) ligases catalyse the formation of the acyl-CoA thioesters of xenobiotic carboxylic acids and the formation of xenobiotic-CoAs has been implicated as being a causative factor in peroxisomal proliferation. In this study we have demonstrated using rat liver peroxisomes that the formation of palmitoyl-CoA is inhibited by a variety of xenobiotic carboxylic acids. Palmitoyl-CoA formation exhibited biphasic kinetics indicative of two isoforms, a high affinity (Km1 2.3 microM) low capacity form and a low affinity (Km2 831 microM) high capacity form. These forms were differentially inhibited by a range of xenobiotics. However, it would appear that the low affinity component may not contribute to any major extent to the formation of xenobiotic-CoAs in vivo. At a concentration of 1 mM, greater than 20% inhibition of the high affinity form was observed with the 2-arylpropionates, ibuprofen, naproxen, benoxaprofen, fenoprofen, indoprofen, ketoprofen, tiaprofenic acid and cicloprofen, the hypolipidaemics, nafenopin and ciprofibrate, and the herbicides, silvex and 2,4,5-trichlorophenoxyacetate. Valproic acid, clofibric acid, salicylic acid and 2,4-dichlorophenoxy-acetate were non-inhibitory at all concentrations studied (0.1-2.5 mM). Analysis of the type of inhibition established that only nafenopin (Ki 430 microM) and ciprofibrate (Ki 97 microM) were competitive inhibitors of palmitoyl-CoA formation suggesting that they bind at the active site and thus potentially function as alternative substrates for the peroxisomal ligase. Notably, clofibric acid which has previously been shown to form clofibroyl-CoA in peroxisomes did not interact with the palmitoyl-CoA ligase thereby suggesting that activation is mediated via an alternative peroxisomal CoA ligase. In addition, the xenobiotic inhibitors of the peroxisomal palmitoyl-CoA ligase differed from those previously reported for the equivalent microsomal enzyme suggesting that the organellar forms may be functionally distinct. This study establishes that numerous xenobiotic carboxylic acids interact with the peroxisomal palmitoyl-CoA ligase; however, it would appear that relatively few function as alternative substrates. The toxicological ramifications of peroxisomally mediated xenobiotic-CoA formation and the identification of other peroxisomal xenobiotic-CoA ligase(s) remain to be elucidated.     

Inhibition kinetics of hepatic microsomal long chain fatty acid-CoA ligase by 2-arylpropionic acid non-steroidal anti-inflammatory drugs.

Microsomal long chain fatty acid CoA ligase (EC 6.2.1.3) has been implicated in the formation of CoA thioesters of xenobiotics containing a carboxylic acid moiety. In this study we have demonstrated that the microsomal enzyme from rat liver exhibits biphasic kinetics for the formation of palmitoyl-CoA, i.e. there are high affinity low capacity Kmhigh, 1.6 microM, Vmaxhigh, 12.9 nmol/mg/min) and low affinity high capacity (Kmlow, 506 microM, Vmaxlow, 58.3 nmol/mg/min) components. Inhibition of the high affinity isoform was studied using the R and S enantiomers of ibuprofen, fenoprofen, ketoprofen and naproxen. The high affinity component of palmitoyl-CoA formation was competitively inhibited by R-fenoprofen (Ki 15.4 microM) while R-ibuprofen exhibited mixed inhibition kinetics. In contrast the R and S enantiomers of ketoprofen and naproxen were non-competitive inhibitors. This diversity of inhibition kinetics observed argues in favour of a binding site in addition to the catalytic site. A competitive interaction with the high affinity form correlated with literature evidence of enantiospecific chiral inversion and "hybrid" triglyceride formation for the R enantiomers of fenoprofen and ibuprofen. Paradoxically, R-ketoprofen which is extensively inverted in rats was a non-competitive inhibitor of palmitoyl-CoA formation by the high affinity isoform suggesting that it may not act as an alternate substrate. The results of this study clearly indicate that formation of R-2-arylpropionate-CoAs is not fully explained by interaction with the high affinity isoform of a microsomal long chain (palmitoyl) CoA ligase and therefore the involvement of other isoforms cannot be discounted.     

Phenothiazines inhibit acetylcholinesterase by concentration-dependent-type kinetics. A study with trifluoperazine and perphenazine.

The properties of perphenazine (PPZ) and trifluoperazine (TFP) as fluorescent dyes were exploited to calculate their critical micellar concentrations. The relative fluorescence quantum yield of the two amphiphiles was dependent on their concentration, abruptly decreasing above 30-40 microM PPZ and 20-30 microM TFP. Evidence is presented that this phenomenon is driven by the formation of non-fluorescent drug aggregates. The type of inhibition kinetics displayed by PPZ and TFP on human erythrocyte acetylcholinesterase (AChE) was also dependent on drug concentration, turning from non-competitive to a "mixed" inhibition type at concentrations at which PPZ and TFP were demonstrated to undergo micelle formation. Results support the notion that phenothiazines may interact with AChE both as monomers and micellar aggregates, producing different inhibitory effects.     

Copper-induced oxidative stress in rainbow trout gill cells

Copper is known to pose a serious threat to aquatic organisms. However, the mechanisms of its toxicity still remain unclear. Cu is known to exert its toxicity partly due to the formation of reactive oxygen species (ROS). The purpose of this work was therefore to link the exposure to copper at pH 6 and 7 to cellular formation of ROS and effects like cell viability and genotoxicity using the rainbow trout gill cell line RTgill-W1. To relate effects to bioavailable copper, free Cu(2+) concentrations in the medium were calculated using the programm ChemEQL 3.0. 2',7'-Dichlorodihydrofluorescein-diacetate (H(2)DCF-DA) was used as cell-permeant indicator of ROS formation. Cell viability was assessed using the fluorogenic probe 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). DNA strand breaks were assessed using the comet assay, and lipid peroxidation was investigated using the thiobarbituric acid-reactive substances assay (TBARS). Copper treatment resulted in a dose-dependent elevation in cytotoxicity and formation of cellular ROS. Cell viability was significantly reduced at total copper (Cu(T)) concentrations of 5 microM (corresponding to a free Cu(2+) of 0.11 microM at pH 7) and higher, resulting in an EC(50) of Cu(T)=29.2 microM (Cu(2+)=0.63 microM, pH 7). Neither an impairment concerning the viability of control cells due to growth at pH 6 was observed nor significant differences for cytotoxicity in cells exposed to the same nominal Cu(T) concentrations at pH 6 compared to pH 7. Cellular ROS concentrations increased significantly and decreased with loss of cell viability. After normalizing ROS formation to cell viability, ROS induction up to 25-35-fold compared to the control was detected, but mainly for rather high concentrations (Cu(T) > or = 100 microM; Cu(2+) > or = 2.2 microM, pH 7). ROS formation rates were slightly higher when cells were exposed to Cu at pH 6 compared to pH 7, correlating with the higher free Cu(2+) concentrations. A significant induction of DNA strand breaks was noted at Cu(T) of 1 and 2.5 microM with greater effects at pH 6 due to higher free Cu(2+) concentrations than at pH 7. No effects on lipid peroxidation were observed. These results lead to the hypothesis that copper-induced loss in viability and genotoxicity in trout gill cells are partially triggered by the generation of ROS and related to the free Cu(2+).     

Drug-S-acyl-glutathione thioesters: synthesis, bioanalytical properties, chemical reactivity, biological formation and degradation

Carboxylic acid-containing drugs can be metabolized to chemically-reactive acyl glucuronide, S-acyl-CoA thioester, and/or intermediate acyl-adenylate metabolites that are capable of transacylating the cysteinyl-thiol of glutathione (GSH) resulting in the formation of drug-S-acyl-GSH thioesters detected in-vivo in bile and in-vitro in hepatocytes. Authentic S-acyl-GSH thioesters of carboxylic acids can be readily synthesized by modifying the cysteinyl-thiol group of GSH with an applicable acylating reagent. Bionanalytical characterization of S-acyl-GSH derivatives has demonstrated enhanced extraction efficiency from biological samples when formic acid is included in appropriate extraction solvents, and that tandem mass spectrometry of S-acyl-GSH conjugates results in fragmentation producing a common MH+-147 Da product ion. Chemical reactivity comparisons have shown that S-acyl-CoA thioester and acyl-adenylate conjugates are more reactive than their corresponding 1-β-O-acyl glucuronides toward the transacylation of GSH forming S-acyl-GSH thioesters. S-Acyl-GSH thioester derivatives are also chemically-reactive electrophiles capable of transacylating biological nucleophiles. Glutathione S-transferases (GSTs) weakly catalyze S-acyl-GSH conjugate formation from S-acyl-CoA, acyl-adenylate, and 1-β-O-acyl glucuronide substrates; however purified-GSTs have also been shown to hydrolyze S-acyl-GSH thioesters. Mechanistic in vitro studies in hepatocytes have revealed the primary importance of the S-acyl-CoA formation pathway leading to S-acyl-GSH-adduct formation. In addition to being hydrolytically-unstable in hepatocytes and plasma, S-acyl-GSH thioesters undergo γ-glutamyltranspeptidase-mediated cleavage of the γ-glutamyl-group leading to N-acyl-cysteinylglycine amide-linked products. In summary, S-acyl GSH thioesters are indicators of reactive transacylating metabolite formation produced from the biotransformation of carboxylic acids, but since they are also chemically-reactive, perhaps these derivatives can contribute to covalent binding to tissue proteins and potential toxicity.     

Fluvoxamine is a more potent inhibitor of lidocaine metabolism than ketoconazole and erythromycin in vitro

CYP3A4 is generally believed to be the major CYP enzyme involved in the biotransformation of lidocaine in man; however, recent in vivo studies suggest that this may not be the case. We have examined the effects of the CYP3A4 inhibitors erythromycin and ketoconazole and the CYP1A2 inhibitor fluvoxamine on the N-deethylation, i.e. formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine by human liver microsomes. The experiments were carried out at lidocaine concentrations of 5 microM (clinically relevant concentration) and 800 microM. The formation of both MEGX and 3-hydroxylidocaine was best described by a two-enzyme model. At 5 microM of lidocaine, fluvoxamine was a potent inhibitor of the formation of MEGX (IC50 1.2 microM). Ketoconazole and erythromycin also showed an inhibitory effect on MEGX formation, but ketoconazole (IC50 8.5 microM) was a much more potent inhibitor than erythromycin (IC50 200 microM). At 800 microM of lidocaine, fluvoxamine (IC50 20.7 microM) and ketoconazole (IC50 20.4 microM) displayed a modest inhibitory effect on MEGX formation, whereas erythromycin was a weak inhibitor (IC50 >250 microM). The 3-hydroxylation of lidocaine was potently inhibited by fluvoxamine at both lidocaine concentrations (IC50 0.16 microM at 5 microM and 1.8 microM at 800 microM). Erythromycin and ketoconazole showed a clear inhibitory effect on the 3-hydroxylation of lidocaine at 5 microM of lidocaine (IC50 9.9 microM and 13.9 microM, respectively), but did not show a consistent effect at 800 microM of lidocaine (IC50 >250 microM and 75.0 microM, respectively). Although further studies are needed to elucidate the role of distinct CYP enzymes in the biotransformation of lidocaine in humans, the findings of this study suggest that while both CYP1A2 and CYP3A4 are involved in the metabolism of lidocaine by human liver microsomes, CYP1A2 is the more important isoform at clinically relevant lidocaine concentrations.     

Protective effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on copper-induced hydroxyl radical generation in the rat heart

The present study was examined the effect of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on Cu(II)-induced hydroxyl radical generation (OH) in the extracellular fluid of rat myocardium. Rats were anesthetized and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. When Cu(II) was infused through the microdialysis probe, Cu(II) increased in OH formation trapped as 2,3-DHBA in the dialysate. When fluvastatin (100 microM) was administered to Cu(II) (50 microM)-pretreated animals, the levels of 2,3-DHBA at 300 min after administration of fluvastatin significantly decreased. In cumulative dose dependent experiments, three concentrations of Cu(II), 10, 25 and 50 microM, were infused through the microdialysis probe in the rat myocardium. A positive linear correlation between Cu(II) and the formation of 2,3-DHBA (R(2)=0.980) was observed. However, when corresponding experiments were performed with fluvastatin (100 microM) pretreated animals, the level of 2,3-DHBA decreased. These results suggest that blocking LDL oxidation by fluvastatin may attenuate Cu(II)-induced OH formation in the rat heart.     

Troglitazone increases cytochrome P-450 3A protein and activity in primary cultures of human hepatocytes.

Troglitazone (TRO) is an insulin sensitizer used in the treatment of type II diabetes. TRO is known to increase the activity of cytochrome P-450 (CYP) 3A in vivo. We have investigated the effect of TRO on CYP3A protein content and the activity of CYP3A (as measured by the formation of 6beta-hydroxytestosterone formation) in primary cultures of human hepatocytes in comparison with rifampicin (RIF). Hepatocytes were isolated from four human livers by perfusion with collagenase, plated on collagen-coated plates, and maintained in William's E medium. After 48 h in culture, cells were exposed to RIF (10 microM) or TRO (0-50 microM) twice, each over a period of 24 h, and the activity of CYP3A was measured. TRO increased the activity of CYP3A in a concentration-dependent manner, reaching a maximal response at 5 microM. Pretreatment of the hepatocytes with 10 microM TRO or 10 microM RIF resulted in a 4- to 15-fold increase in the activity of CYP3A. Maximum increase in CYP3A protein was observed at 5 microM TRO. There was a significant correlation (R(2) = 0.89) between the content of immunoreactive CYP3A protein in the hepatocytes and the rate of formation of 6beta-hydroxytestosterone. These results indicate that TRO is a potent inducer of CYP3A and is similar to RIF in inducing CYP3A in human hepatocytes. At concentrations of 25 microM and above, TRO was toxic to the cells, as determined by a decrease in the activity of CYP3A, a reduction in the amount of immunoreactive protein, and changes in the morphology of the cells.     

Cytochrome P450 enzymes contributing to demethylation of maprotiline in man

From case reports of patients treated with the tetracyclic antidepressant drug maprotiline, it appears that this drug is subject to polymorphic metabolism. Thus, we studied formation of the major maprotiline metabolite desmethylmaprotiline to identify the human cytochrome P-450 enzymes (CYP) involved. In incubations with human liver microsomes from two different donors, the substrate maprotiline was used at five different concentrations (5 to 500 microM). For selective inhibition of CYPs, quinidine (0.5-50 microM; CYP2D6), furafylline (0.3-30 microM; CYP1A2), ketoconazole (0.2-20 microM; CYP3A4), mephenytoin (20-200 microM; CYP2C19), chlorzoxazone (1-100 microM; CYP2E1), sulphaphenazole (0.2-100 microM; CYP2C9) and coumarin (0.2-100 microM; CYP2A6) were used. Desmethylmaprotiline concentrations were measured by HPLC, and enzyme kinetic parameters were estimated using extended Michaelis-Menten equations with non-linear regression. Relevant inhibition of the desmethylmaprotiline formation rate was observed in incubations with quinidine, furafylline and ketoconazole only. Formation rates of desmethylmaprotiline were consistent with a two enzyme model with a high (K(M)=71 and 84 microM) and a low (K(M)=531 and 426 microM) affinity site for maprotiline in the two samples, respectively. The high affinity site was competitively inhibited by quinidine (K(i,nc) 0.13 and 0.61 microM), the low-affinity site was non-competitively inhibited by furafylline (K(i,nc) 0.11 and 1.3 microM). Thus it appears that CYP2D6 and CYPIA2 contribute to maprotiline demethylation. Based on the parameters obtained, for plasma concentrations of 1 microM 83% (mean) of desmethylmaprotiline formation in vivo is expected to be mediated by CYP2D6 while 17% only may be attributed to CYPIA2 activity.     

Inhibitory effect of various thrombin inhibitors on shear-induced platelet function and dynamic coagulation

We assessed the effects of active site-directed, fibrinogen recognition exosite (FRE)-directed and bifunctional thrombin inhibitors, on shear-induced platelet reactivity (adhesion/aggregation) and dynamic coagulation (coagulation of flowing blood). An in vitro test for shear-induced haemostatic plug formation and dynamic coagulation (haemostatometry) was employed using non-anticoagulated rat blood. The active site-directed inhibitors (argatroban, P891, P899) caused inhibition of platelet reactivity and coagulation at 1-, 100- and 100-microM concentrations, respectively. Bifunctional inhibitors (P553, P1053) exerted inhibitory effects at 0.1 microM. A dimeric bifunctional inhibitor P824 caused significant inhibition at 1 microM. The FRE-directed inhibitor (P960) inhibited shear-induced platelet reactivity at 10 microM but the dynamic coagulation at 1 microM. Combination of active site-directed argatroban and FRE-directed P960 did not show any synergistic effect. The most potent inhibition was observed in monomeric bifunctional inhibitors. The inhibitory effects were compared with the K(i) values against human thrombin and with the IC(50) values against fibrin clot formation. The minimum effective concentrations on shear-induced platelet reactivity and dynamic coagulation were comparable with the IC(50) values, but not with the K(i) values.     

D2-dopamine receptor-mediated inhibition of cyclic AMP formation in striatal neurons in primary culture

Dopamine (DA) regulation of intracellular cyclic AMP formation in purified, intact striatal neurons in primary culture was examined. DA (EC50, 3 microM) and vasoactive intestinal polypeptide (VIP; EC50, 10 nM) stimulated cyclic AMP formation by 2- and 5-fold, respectively. In the presence of 0.1 microM forskolin (which was virtually ineffective alone), neurohormone efficacy was augmented; potency was unaffected. In the presence of 0.1 microM SCH 23390, a selective D1 antagonist, the DA dose-response curve was shifted rightward in a competitive manner. At low concentrations (0.01-1.0 microM), however, DA inhibited basal cyclic AMP formation. The inhibitory effect, but not the shift of the dose-response curve, was blocked by 5 microM l-sulpiride, a selective D2 antagonist. At saturating concentrations of VIP (0.1-1.0 microM), no other neurohormone can further augment cyclic AMP formation. Under these conditions, increasing concentrations of DA resulted in a dose-dependent (IC50, 0.5 microM) inhibition of VIP-stimulated cyclic AMP synthesis. This effect was augmented in the presence of 0.1 microM SCH 23390 and blocked by 5 microM l-sulpiride. Sulpiride antagonism was stereospecific, with the l-isomer being 30-fold more potent than the d-isomer. The rank order of potency for a series of dopaminergic agonists and antagonists at the receptor mediating attenuation of cyclic AMP formation suggests that it is of the D2 type. Furthermore, both DA and Met-enkephalin inhibition of cyclic AMP formation is lost after exposure of striatal neurons to islet activator protein. These findings suggest that a D2 receptor mediates the inhibition of intracellular cyclic AMP formation by DA in striatal neurons in primary culture, and may do so by an interaction with the inhibitory guanine nucleotide regulatory protein of adenylate cyclase.     

CYP3A4 activity in four different animal species liver microsomes using 7-benzyloxyquinoline and HPLC/spectrofluorometric determination

Some microplate-based direct assays with different fluorometric substrates have been developed, among which 7-benzyloxyquinoline (BOQ) has demonstrated the highest degree of selectivity for CYP3A subfamily. In our study, we firstly developed and validated an efficient, fast and cheap HPLC/spectrofluorometric analytical method to quantify 7-hydroxyquinoline (BOQ metabolite). Secondly, BOQ oxidation rate (1.95 +/- 0.24 microM/mg protein/min) was compared to that of midazolam (MDZ) (1.4 +/- 0.21 microM/mg protein/min), an other specific CYP3A probe. However, the difference did not reach statistically significance (test of Sign; p = 0.125, two tailed). Thirdly, the potential use of BOQ in other species than the rat (mouse, dog and monkey) was studied. The highest BOQ activity was observed in rat microsomes (3.75 micromol/mg protein/min) with lower P450 content (0.3 nmol/mg protein) compared to other species. Finally, the effect of CYP3A enzymes-selective inhibitor ketoconazole on the dealkylation of BOQ in control and dexamethasone (DM)-treated rat microsomes was studied. Ketoconazole inhibition potency was greater in control (IC(50) approximately 21.6 microM) compared to DM induced (IC(50) approximately 32.3 microM) microsomes. At concentrations greater than that considered to be enzyme-selective (e.g., 10-30 microM), ketoconazole inhibitory activity did not rise significantly, and at the maximal concentration tested (1,000 microM) a nearly similar inhibition (76%) was observed than that at 50 microM concentration (68.2%).     

Effect of fibrinopeptides A and B on human and rat platelet aggregation in vitro

The effect of fibrinopeptides on platelet aggregation is reported. Fibrinopeptide A (minimal effective concentration, 0.65 microM) aggregated human (but not rat) platelets suspended in plasma and at lower concentrations (0.01-0.1 microM) potentiated platelet aggregation due to ADP and collagen in both species. Fibrinogen mimicked these effects of fibrinopeptide A. P-bromophenacyl bromide (100 microM), mepacrine (10 microM), indomethacin (10 microM) and dazoxiben (10 microM) inhibited human platelet aggregation induced by fibrinopeptide A and fibrinogen. In both species, fibrinopeptide B (0.65-6.5 microM) antagonised the platelet inhibitory effect of PGI2 and PGD2 but not adenosine. Antagonism was non-competitive in nature. The concentration of fibrinopeptide A required to potentiate platelet aggregation occurs naturally in the plasma of patients with thrombotic disease suggesting this effect may be of physiological significance during the formation of a thrombus. The novel action of fibrinopeptide B to reduce the platelet inhibitory effect of PGI2 and PGD2 may also contribute to the control of thrombus formation.     

Possible role of dimethylarsinous acid in dimethylarsinic acid-induced urothelial toxicity and regeneration in the rat

Dimethylarsinic acid (DMA(V)) is carcinogenic to the rat urinary bladder when administered at high doses in the diet or drinking water. At a dietary dose of 100 ppm (microg/g), it produces cytotoxicity within 6 h and increased proliferation (hyperplasia) by 7 days of administration. We hypothesize that formation of the reactive organic intermediate dimethylarsinous acid (DMA(III)) is involved in the induction of the cytotoxicity. To evaluate the possibility that DMA(V) administration produces urothelial toxicity and regeneration by the formation of trivalent arsenicals, 2,3-dimercaptopropane-1-sulfonic acid (DMPS, 5600 ppm), a chelator of trivalent arsenicals, was co-administered with DMA(V) (100 ppm) for 2 weeks to groups of female Fischer F344 rats. Based on light and scanning electron microscopy, and bromodeoxyuridine labeling index, DMA(V) produced cytotoxicity and regenerative hyperplasia of the urothelium which was inhibited by co-administration with DMPS. The major forms of arsenic in the 24-h urine of rats administered DMA(V) were high concentrations of DMA(V) (66.4 +/- 2.7 microM) itself and the pentavalent organic arsenical trimethylarsine oxide (TMAO) (73.2 +/- 9.5 microM). Co-administration with DMPS led to an increase in DMA(V) (507 +/- 31 microM) with a decrease in TMAO (2.8 +/- 0.4 microM) excretion. The formation of TMAO from DMA(V) mechanistically suggests formation of the intermediate trivalent metabolite, DMA(III). In a second experiment evaluating fresh void urines collected on study days 1, 71, and 175, we detected DMA(III) in the urine of DMA(V) and DMA(V) plus DMPS-treated rats at approximately micromolar concentrations. Using rat (MYP3) and human (1T1) urothelial cells, cytotoxicity for trivalent arsenicals, sodium arsenite, monomethylarsonous acid (MMA(III)), and DMA(III) was demonstrated at 0.4-4.8 microM concentrations, whereas MMA(V), DMA(V), and TMAO were cytotoxic at millimolar concentrations. The presence of DMA(III) at micromolar concentrations in the urine of rats fed 100 ppm DMA(V) suggests that DMA(III) produced in vivo may be involved in the toxic effects in the rat urinary bladder after dietary administration of DMA(V).     

Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells.

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.     

Triazolam substrate inhibition: evidence of competition for heme-bound reactive oxygen within the CYP3A4 active site.

In human liver microsomes, triazolam is principally metabolized by CYP3A4 to form two metabolites, 1'-hydroxytriazolam (1'OHTz) and 4-hydroxytriazolam (4OHTz). The velocity of 1'OHTz formation was found to decrease at higher triazolam concentrations (>200 microM), indicative of "substrate inhibition". Coincubation of [(14)C]triazolam with authentic metabolite standards of either 1'OHTz or 4OHTz up to 30 microM did not significantly inhibit the rate of [(14)C]1'OHTz formation. The effects of secondary compounds on triazolam oxidation were shown to be product-specific, producing either activation or inhibition depending on the triazolam metabolite monitored. When human liver microsomes were supplemented with exogenous human cytochrome b(5), it was observed that substrate inhibition was attenuated and the resulting increase in 1'OHTz formation, relative to control (nonsupplemented) incubations, corresponded to a decrease in the ratio of 4OHTz to 1'OHTz. In contrast, when cofactor (e.g., 100 microM NADPH) was rate limiting, the metabolite ratio (4OHTz/1'OHTz) was markedly increased over the entire substrate concentration range (0.5-1000 microM). To explain these kinetic observations, a two-site binding model is proposed in which triazolam is hypothesized to bind within the CYP3A4 active site in spatially distinct orientations, which may lead to the formation of either the 1'-hydroxytriazolam or 4-hydroxytriazolam. Differential inhibition/activation is consistent with this two-site model and substrate inhibition is hypothesized to result from competition between the two sites for reactive oxygen.     

Involvement of CYP1A2 and CYP3A4 in lidocaine N-deethylation and 3-hydroxylation in humans.

The roles of cytochrome P-450 (CYP) enzymes in the N-deethylation, i.e., formation of monoethylglycinexylidide (MEGX), and 3-hydroxylation of lidocaine were studied with human liver microsomes and recombinant human CYP isoforms. Both CYP1A2 and CYP3A4 were found to be capable of catalyzing the formation of MEGX and 3-OH-lidocaine. Lidocaine N-deethylation by liver microsomes was strongly inhibited by furafylline (by about 60%) and anti-CYP1A1/2 antibodies (>75%) at 5 microM lidocaine, suggesting that CYP1A2 was the major isoform catalyzing lidocaine N-deethylation at low (therapeutically relevant) lidocaine concentrations. Troleandomycin inhibited the N-deethylation of lidocaine by about 50% at 800 microM lidocaine, suggesting that the role of CYP3A4 may be more important than that of CYP1A2 at high lidocaine concentrations. Chemical inhibition and immunoinhibition studies also indicated that 3-OH-lidocaine formation was catalyzed almost exclusively by CYP1A2, CYP3A4 playing only a minor role. Although the CYP2C9 inhibitor sulfaphenazole (100 microM) inhibited MEGX formation by about 30%, recombinant human CYP2C9 showed very low catalytic activity, suggesting a negligible role for this enzyme in lidocaine N-deethylation. Chemical inhibition studies indicated that CYP2C19, CYP2D6, and CYP2E1 did not play significant roles in the metabolism of lidocaine in vitro. Taken together, these results demonstrate that CYP1A2 and CYP3A4 enzymes are the major CYP isoforms involved in lidocaine N-deethylation. Therefore, the MEGX test (formation of MEGX from lidocaine) is not a suitable marker of hepatic CYP3A4 activity in vivo.     

The effect of vindesine on methotrexate hydroxylation in the rat

The effect of vindesine (VDS) on methotrexate (MTX) disposition was studied in bile-drained rats administered VDS prior to [3H]MTX, and in isolated rat hepatocytes and rat liver homogenate concomitantly incubated with MTX and VDS at 37 degrees. In vivo, 7-hydroxylation was reduced by 0.65 mg/kg VDS. In VDS-treated animals, biliary recovery of the MTX dose (50 mg/kg) as 7-hydroxymethotrexate (7-OH-MTX) (1.75 +/- 0.2%, mean +/- SEM) was significantly reduced compared to controls (2.83 +/- 0.57%). In vitro, hydroxylation of MTX (10-200 microM) in hepatocytes was reduced by 14.3 and 66.4% (means) at 12.5 and 100 microM VDS, respectively. With increasing VDS concentrations up to 100 microM, a reduction in intracellular MTX accumulation could account for the decreased MTX hydroxylation. Experiments in a cell free system gave no evidence of inhibition of 7-OH-MTX formation by VDS. In vitro MTX transport studies demonstrated that VDS inhibited the hepatocellular influx of MTX, as (1) the accumulation of MTX corresponded inversely to increasing VDS concentrations and (2) the MTX efflux was not increased by VDS. The apparent Ki for VDS inhibition of MTX influx was 57 microM. We suggest that VDS, by reducing the 7-OH-MTX formation in liver cells, may have implications for combination chemotherapy regimens which include MTX.